CN105838633A

CN105838633A - Mycobacterium foruitum and application thereof

Info

Publication number: CN105838633A
Application number: CN201510016326.8A
Authority: CN
Inventors: 薛建萍; 姚再男; 唐璐敏; 范锦锦; 朱健; 丁林富; 卜桂琴
Original assignee: Shanghai Pesticide Research Institute Co Ltd
Current assignee: Shanghai Pesticide Research Institute Co Ltd
Priority date: 2015-01-13
Filing date: 2015-01-13
Publication date: 2016-08-10

Abstract

The invention relates to the fields of industrial microorganisms and fermentation technology, specifically, to mycobacterium foruitum having a catalytic function and an application of mycobacterium foruitum. The mycobacterium foruitum is provided with the accession number CGMCC No.9657, and can catalyze phytosterol to produce 9[alpha]-hydroxylandrostenedione. The conversation rate is over 95%, and the selectivity is 50-60%. The unit consumption in the production of 9[alpha]-hydroxylandrostenedione through utilization of mycobacterium foruitum is 2.2-2.5, and the main product is 9[alpha]-hydroxylandrostenedione. The preparation method is simple, is high in yield, and has bright application prospects.

Description

A kind of Mycobacterium fortuitum and application thereof

Technical field

The invention belongs to biotechnology and biological chemical field, specially industrial microorganism and fermentology neck Territory, a kind of Mycobacterium fortuitum with catalysis and application thereof.

Background technology

Steroid hormone class medicine refers in molecular structure containing the hormone medicine of steroidal structure, is a class clinically Important medicine, has the pharmacological actions such as the strongest infection, antiallergic, antiviral and shock, is medicine One of prior development direction of industry.This kind of drug main adrenocortical hormone to be included and the big class of gonadal hormone two. Corticosteroids medicine has cortisone acetate (cortisone acetate), hydrocortisone for clinical (hydrocortisone), dexamethasone acetate (oexamethasone acetate), fluocinonide (fluocinonide) Deng.

The important source material of steroid hormone class drug manufacture is plant sterol.Convert through mycobacterium from plant sterol Generate 9 Alpha-hydroxies-androstenedione (also known as 9-hydroxyl-androstenedione, 9OH-AD, 9 α-OH AD).9 α-hydroxyl Base-androstenedione can be as the synthesis material of multiple important steroidal drug.

The method complex manufacturing productivity of chemosynthesis is the highest, and therefore bioprocess technology fermentation process produces 9 α-hydroxyl Base-androstenedione is developing direction.

In existing report, new golden mycobacteria (M ycobacterium sp.) microorganism catalysis is used to plant Thing sterol is converted into androstenedione, i.e. 4-AD (AD) or androstane-Isosorbide-5-Nitrae-alkene-3,17-diketone (ADD), by mutagenesis screening to bacterial strain after ADD yield can significantly improve.Androstenedione need to pass through into Single step reaction generates 9 Alpha-hydroxies-androstenedione.

The invention provides a kind of Mycobacterium fortuitum, plant sterol can be catalyzed and be converted into 9 Alpha-hydroxies-androstene two Ketone.

Summary of the invention

It is an object of the invention to provide a kind of new Mycobacterium fortuitum strain.This bacterial strain has catalysis plant Sterol produces the function of 9 Alpha-hydroxy androstenedione.

Further object is that employing above-mentioned bacterial strains produces 9 Alpha-hydroxy androstenedione.

Technical scheme is, a kind of Mycobacterium fortuitum (latin name Mycobacterium foruitum, classification life Name: Mycobacterium fortuitum), preserving number is CGMCC No.9657 (preservation mechanism: Chinese microorganism strain Preservation administration committee common micro-organisms center；Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in Institute of microbiology of academy of science of state；Preservation date JIUYUE in 2014 15 days)

The condition of culture of this bacterial strain is: cultivate 6-7 days for 28 DEG C on slant medium.Slant medium can make By the culture medium that Mycobacterium fortuitum is conventional, typically, its component is: Carnis Bovis seu Bubali cream 0.2%, peptone 0.4%, Glycerol 2%, agar powder 2% (pH is natural).

Bacterial strain storage conditions is vacuum lyophilization (long term storage).

This Mycobacterium fortuitum strain can be used for being catalyzed plant sterol and is converted into 9 Alpha-hydroxy androstenedione.

The preparation technology of a kind of 9 Alpha-hydroxy androstenedione, with plant sterol as raw material, with above-mentioned occasional branching stem bar Bacterium microbe conversion, the condition of fermentation is: at 25～34 DEG C, (preferably 26～32 DEG C) are passed through air and stir； Ventilation ratio is 1:0.3～0.85, microbe conversion 48～100 hours.

Preferably ventilation ratio is 1:0.5～0.75；More preferably 1:0.55～0.65.Preferably mixing speed is 300～600rpm, more preferably 450～500rpm.Preferably fermentation time is 48～120 hours, more preferably It it is 72～96 hours.

The condition that preferred microbe conversion is cultivated is: temperature 29 ± 1 DEG C, ventilation ratio 1:0.6, mixing speed 480rpm, the microbe conversion time is 72～96 hours.

The formula of microbe conversion culture medium contains: (a) glycerol 0.5%～2%, (b) sodium phosphate or acid phosphate Sodium salt 0.75%～0.9%, and potassium phosphate or acid phosphate potassium salt 0.35%～0.5%, (d) ammonium salt such as NH₄Cl 0.15%～0.3%, (e) MgSO₄0.02%～0.06%, (f) analysis for soybean powder 0.05%～0.2% or 0.5%～ The peptone of 1% or yeast extract；(g) dispersant (such as Tween 80) 0.05%～0.2%；It is percent mass Ratio.

Possibly together with plant sterol 0.7%～2% in microbe conversion culture medium.Surplus is water.

It is furthermore preferred that the consisting of of microbe conversion culture medium: glycerol 1%, Na₂HPO₄0.84%, KH₂PO₄ 0.45%, NH₄Cl0.2%, MgSO₄0.03%, analysis for soybean powder 0.1%, plant sterol 1%, dispersant is such as told Temperature 80 0.1%, the rest is water, pH 8.5.

Preferably, first Mycobacterium fortuitum is inoculated in seed culture medium, 25～34 DEG C (preferably 26～ 32 DEG C) under be passed through air and stir, be inoculated in fermentation culture after cultivating 48～120 hours.Ventilation ratio is 1:0.3～0.85, preferably 1:0.5～0.75, mixing speed is 300～600rpm, preferably 450～500rpm. Take the volume ratio inoculation fermentation culture fluid that seed culture fluid is with 5%～12%, preferably 10%.

The seed culture medium used in fermentation contains: peptone 0.8%～1.4%, Carnis Bovis seu Bubali cream 0.2%～0.5%, NaCl0.45%～0.6% (is mass percent), pH6.8～7.3.Preferably, seed culture medium pH=6.9～ 7.0, containing peptone 1%, Carnis Bovis seu Bubali cream 0.3% and NaCl0.5%.

Preferably, seed culture condition is: temperature 29 ± 1 DEG C, ventilation ratio 1:0.4～1:0.75, rotating speed 300～ 600rpm, cultivates 72-96 hour；It is furthermore preferred that seed culture condition is: temperature 29 ± 1 DEG C, ventilation ratio 1:0.6, rotating speed 480rpm, cultivate 72-96 hour.

Fermentation liquid organic solvent, such as ethyl acetate, dichloromethane or dichloroethane extraction.Fermentation liquid is with organic Solvent volume is than for 1:2～2:1, preferably 1:1.2～1.2:1.

After testing, the primary product with the Mycobacterium fortuitum microbe conversion plant sterol of the present invention is 9 Alpha-hydroxies Androstenedione, conversion ratio more than 95%, selectivity reaches 50%～60%, produces the list of 9 Alpha-hydroxy androstenedione Consumption is 2.2～2.5.

Mycobacterium fortuitum provided by the present invention, the primary product of catalyzed conversion plant sterol is that 9 Alpha-hydroxies are male Alkene diketone, preparation method is simple, and efficiency is high, has good application prospect.

Accompanying drawing explanation

Fig. 1 is in embodiment 2, after fermenting 96 hours, extracts the liquid chromatograph (HPLC) of fermentation liquid detection Figure

Wherein, a by-product 1；B ethyl acetate；C 9 Alpha-hydroxy androstenedione；D 9 Alpha-hydroxy testosterone (by-product 2)；E. by-product 3；F by-product 4

Fig. 2 is in embodiment 2,9 Alpha-hydroxy androstenedione content in different fermentations time fermentation liquid.

Specific embodiments

Embodiment 1

The wild type Mycobacterium fortuitum of the present invention, gathers from soil.In August, 2011 is collected in Hunan Zhang Jia Boundary.

Latin name: Mycobacterium foruitum

Preserving number: CGMCC No.9657

Preservation date: on JIUYUE 15th, 2014.

Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC).

Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.

The storage conditions of above-mentioned Mycobacterium fortuitum is: vacuum lyophilization (long term storage).

Condition of culture is: can be by general accidental mycobacterium culture medium, 25～30 DEG C (preferably 28 DEG C) Slant culture 6～7 days in Fructus Solani melongenae bottle.Typical slant culture based component: Carnis Bovis seu Bubali cream 0.2%, peptone 0.4%, glycerol 2%, agar powder 2% (pH is natural).

Embodiment 2

Above-mentioned Mycobacterium fortuitum can be catalyzed plant sterol and produce 9 Alpha-hydroxy androstenedione.

20L seed culture medium is positioned in 35L seed tank, accesses the bacterium (inclined-plane on a Fructus Solani melongenae bottle inclined-plane Area is about 8cm × 8cm).Seed culture medium contains peptone 1%, Carnis Bovis seu Bubali cream 0.3%, and NaCl0.5% is (all For mass percent), and pH is adjusted to 7.0.

Seed culture condition is: temperature 29 ± 1 DEG C, and ventilation ratio is 1:0.6, rotating speed 480rpm, cultivates 96 Hour.

Take seed culture medium, be inoculated in fermentation culture by 10% volume ratio, be placed in fermentation in 50 liters of fermentation tanks (charging 30L), for transformation phytosterin.The pH=8.5 of fermentation culture, and contain: analysis for soybean powder 0.1%, Glycerol 1%, Na₂HPO₄0.84%, KH₂PO₄0.45%, NH₄Cl0.2%, MgSO₄0.03%, plant Sterol 1%, Tween 80 0.1% (being mass percent), surplus is water.

The condition that microbe conversion is cultivated is: temperature 29 ± 1 DEG C, ventilation ratio 1:0.6, rotating speed 480rpm.

Transformation time takes fermentation liquid for 48,72,96,120 hours respectively, and extracts by equal-volume ethyl acetate. With the content of high performance liquid chromatography detection ethyl acetate middle product mutually, in every milliliter of ethyl acetate, 9 Alpha-hydroxies are male The content of alkene diketone is as in figure 2 it is shown, fermentation time is the highest in 72～96 hourly outputs.

Ferment and within 96 hours, take the extraction of fermentation liquid equal-volume ethyl acetate, detect acetic acid second by high performance liquid chromatography Ester mutually in the content of each product and the surplus (with standard substance as work standard specimen) of plant sterol.Microbe conversion Primary product be 9 Alpha-hydroxy androstenedione (9 α-OH AD), by-product is mainly 9 Alpha-hydroxy testosterone (Fig. 1 In d)；9 Alpha-hydroxy androstenedione and 9 Alpha-hydroxies testosterone production ratio (mol ratio) are at 3:1 to 5:1.Turn Rate reaches more than 95%, and the unit consumption producing 9 Alpha-hydroxy androstenedione is 2.2～2.5, and selectivity 50%～ 60%.

Conversion ratio %=conversion of substrate amount ÷ puts into amount of substrate × 100

Yield %=actual product amount ÷ theoretical product amount × 100

Selectivity %=yield ÷ conversion ratio × 100

Unit consumption=actual product amount ÷ puts into amount of substrate

Above substrate is plant sterol, and product is 9 Alpha-hydroxy androstenedione；Conversion of substrate amount=input amount of substrate- Residue amount of substrate；Theoretical product amount=input amount of substrate ÷ substrate molecule amount × molecular weight of product, substrate mean molecule Amount 414, molecular weight of product 302.

Claims

1. a Mycobacterium fortuitum, it is characterised in that preserving number is CGMCC No.9657.

2. the application in producing 9 Alpha-hydroxy androstenedione of the Mycobacterium fortuitum described in claim 1.

3. the Mycobacterium fortuitum described in claim 1 is used for being catalyzed plant sterol and is converted into 9 Alpha-hydroxy androstenes Diketone.

4. the method preparing 9 Alpha-hydroxy androstenedione, it is characterised in that with plant sterol for initial former Material, ferments with the Mycobacterium fortuitum described in claim 1 and is catalyzed, generate 9 Alpha-hydroxy androstenedione.

5. the method preparing 9 Alpha-hydroxy androstenedione described in claim 4, it is characterised in that fermentation condition For: the Mycobacterium fortuitum described in claim 1 is inoculated in the fermentation culture containing plant sterol, It is passed through air at 25～34 DEG C and stirs；The microbe conversion time is 48～100 hours.

6. the method preparing 9 Alpha-hydroxy androstenedione described in claim 5, it is characterised in that during microbe conversion Between be 72～96 hours.

7. the method preparing 9 Alpha-hydroxy androstenedione described in claim 5, it is characterised in that by occasional branching stem Bacillus is inoculated in seed culture medium, is passed through air and stirs at 25～34 DEG C, after cultivating 48～120 hours again It is inoculated in fermentation culture.

8. the method preparing 9 Alpha-hydroxy androstenedione described in claim 5 or 7, it is characterised in that fermentation training Nutrient solution pH=8.3～8.7, containing the material of following mass percent: analysis for soybean powder 0.05%～0.2%, glycerol 0.5%～2%, Na₂HPO₄0.75%～0.9%, KH₂PO₄0.35%～0.5%, NH₄Cl 0.15%～ 0.3%, MgSO₄0.02%～0.06%, plant sterol 0.7%～2%, dispersant 0.05%～0.2%；

Seed culture medium pH=6.8～7.3, containing the material of following mass percent: peptone 0.8%～1.4%, Carnis Bovis seu Bubali cream 0.2%～0.5%, NaCl 0.45%～0.6%.

9. the method preparing 9 Alpha-hydroxy androstenedione described in claim 5 or 7, it is characterised in that seed Ventilation ratio when cultivating and ferment is 1:0.3～0.85, and mixing speed is 300～600rpm.

10. the method preparing 9 Alpha-hydroxy androstenedione described in claim 5 or 7, it is characterised in that ventilation Ratio is 1:0.5～0.75, and mixing speed is 450～500rpm.